Methods of inducing or enhancing cartilage repair

ABSTRACT

The invention relates to methods of enhancing repair of a cartilage and/or inducing formation of a cartilage by administering a cell, which expresses a factor of the T-box family, which includes inter-alia the brachyury. In another embodiment, the invention relates to an engineered cell, which is transfected with a vector comprising a nucleic acid sequence encoding a factor of the T-box family, thereby expressing a factor of the T-box family. In another embodiment, the invention relates to compositions comprising a vector, which comprises a nucleic acid sequence encoding a factor of the T-box family and in another embodiment the composition-comprising cell that expresses a factor of the T-box family, which includes inter-alia the brachyury.

[0001] This application is a Continuation-in Part Application of U.S.Ser. No. 10/067,980, filed Feb. 8, 2002, which claims the benefit ofU.S. Ser. No. 09/376,276, filed Aug. 2, 1999, now abandoned, whichclaims priority from DE Application No. 198.37.438.0, filed Aug. 18,1998, which are incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The invention provides methods of enhancing repair of a cartilageand/or inducing formation of a cartilage by contacting a cell, whichexpresses at least one factor of the T-box family. In anotherembodiment, the invention provides an engineered cell, which istransfected with a vector comprising at least one nucleic acid sequenceencoding a factor of the T-box family, thereby expressing at least onefactor of the T-box family. This invention provides a compositioncomprising a vector, which comprises at least one nucleic acid sequenceencoding a factor of the T-box family.

BACKGROUND OF THE INVENTION

[0003] The meniscus, fibrocartilaginous tissue found within the kneejoint, is responsible for shock absorption, load transmission, andstability within the knee joint. According to the National Center forHealth Statistics, over 600,000 surgeries each year are the result ofcomplications with the meniscus. The meniscus has the intrinsic abilityto heal itself; unfortunately, this property is limited only to thevascular portions of the tissue. For damage outside of these areas andoverall degeneration of the tissue, methods need to be developed thatwill assist the meniscus in healing itself Sweigart MA Tissue Eng 7(2),111-29 (April 2001).

[0004] Degeneration of articular cartilage in osteoarthritis is aserious medical problem caused by arthritis, both rheumatoid andosteoarthritis. Drugs are given to control the pain and to keep theswelling down, but the cartilage continues to be destroyed. Eventually,the joint must be replaced. It is still unknown why cartilage does notheal and no solutions to this problem are known Mankin, N. E. J. Med.331(14), 940-941 (October 1994). Soon after superficial injury,chondrocytes adjacent to the injured surfaces show a brief burst ofmitotic activity associated with an increase in glycosaminoglycan andcollagen synthesis. Despite these attempts at repair, there is noappreciable increase in the bulk of cartilage matrix and the self-repairprocess is usually ineffective in healing the defects.

[0005] Osteochondral, or full-thickness, cartilage defects expand intothe subchondral bone. Such defects arise after the detachment ofosteochondritic dissecting flaps, fractured osteochonidial fragments, orfrom chronic wear of degenerative articular cartilage. Osteochondraldefects depend on the extrinsic mechanism for repair. Extrinsic healingrelies on mesenchymal elements from subchondral bone to participate inthe formation of new connective tissue. This fibrous tissue may or maynot undergo metaplastic changes to form fibrocartilage. Even iffibrocartilage is formed, it does not display the same biochemicalcomposition or mechanical properties of normal articular cartilage orsubchondral bone and degenerates with use, Furukawa, et al., J. BoneJoint Surg. 62A, 79 (1980); Coletti, et al., J. Bone Joint Surg. 54A,147 (1972); Buckwalter, et al., “Articular cartilage: composition,structure, response to injury and methods of facilitating repair”, inArticular Cartilage and Knee Joint Function: Basic Science andArthroscopy, Ewing J E, Ed., (New York, Raven Press, 1990), 19.

[0006] Injection of dissociated chondrocytes directly into the site ofthe defect has also been described as a means for forming new cartilage,as reported by Brittberg, et al., N.E.J. Med. 31, 889-895 (October1994). Cartilage was harvested from minor load-bearing regions on theupper medial femoral condyle of the damaged knee, cultured, andimplanted two to three weeks after harvesting.

[0007] Moreover, if the defect includes a part of the underlying bone,this is not corrected by the use of chondrocytes. The bone is requiredto support the new cartilage.

[0008] Cartilage grafts are also needed in plastic surgery like inrhinoplasty, and the reconstruction of ears.

[0009] The possibility of using stem cells was also examined. Stem cellsare cells which are not terminally differentiated, which can dividewithout limit, and divide to yield cells that are either stem cells orwhich irreversibly differentiate to yield a new type of cell.Unfortunately, there is no known specific inducer of the mesenchymalstem cells that yields only cartilage. In vitro studies in whichdifferentiation is achieved using different bioactive factors ormolecules, yields differentiation of the cells to cartilage whicheventually calcified and turned into bone.

[0010] Thus, there is a need to have a method and composition for theformation or repair of a cartilage or a bone. In another embodiment, itwill be highly advantageous to have a cell, which can divide and form acartilage or a bone tissue.

SUMMARY OF THE INVENTION

[0011] In one embodiment the invention provides a method of enhancingrepair of a cartilage comprising the step of administering to a subjectan effective amount of a cell which expresses at least one factor of theT-box family, thereby enhancing repair of the cartilage.

[0012] In another embodiment the invention provides a method of inducingformation of a cartilage comprising the step of administering to asubject an effective amount of a cell which expresses at least onefactor of the T-box family, thereby inducing formation of the cartilage.

[0013] In another embodiment the invention provides a method ofenhancing repair of a cartilage in the body comprising the step ofadministrating a recombinant vector which comprises a nucleic acidencoding a factor of the T-box family to the cartilage of a subject,thereby enhancing repair of the cartilage.

[0014] In another embodiment the invention provides a method of inducingformation of a cartilage in the body comprising the step ofadministrating a recombinant vector which comprises a nucleic acidencoding a factor of the T-box family to the cartilage of a subject,thereby inducing formation of the cartilage.

[0015] In another embodiment the invention provides a method of inducingchondrocyte differentiation comprising the step of administering of arecombinant vector, which comprises a nucleic acid encoding a factor ofthe T-box family, thereby inducing chondrocyte formation.

[0016] In another embodiment the invention provides a method ofrepairing or forming a cartilage in a subject in need comprising thesteps of: obtaining a cell from of the subject; transfecting said cellwith a recombinant vector comprising a nucleic acid sequence encoding afactor of the T-box family, so as to obtain an engineered cell whichexpresses a factor of the T-box family; and administering saidengineered cell to the subject.

[0017] In another embodiment the invention provides a method for theproduction of transplantable cartilage matrix, the method comprising thesteps of: obtaining a cell; transfecting said cell with a recombinantvector comprising a nucleic acid sequence encoding a factor of the T-boxfamily, so as to obtain an engineered cell which expresses a factor ofthe T-box family; and culturing said cell with the cell-associatedmatrix for a time effective for allowing formation of a transplantablecartilage matrix.

[0018] In another embodiment the invention provides an engineered cell,which expresses a factor of the T-box family.

[0019] In another embodiment the invention provides an implant devicecomprising at least one engineered cell which expresses a factor of theT-box family and a pharmaceutically acceptable carrier.

[0020] In another embodiment the invention provides a compositioncomprising an engineered cell which expresses a factor of the T-boxfamily and a pharmaceutically acceptable carrier.

[0021] In another embodiment the invention provides a compositioncomprising at least one recombinant vector which comprises a nucleicacid sequence encoding at least one factor of the T-box family and apharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022]FIG. 1. FGFR3 mediates chondrocytic differentiation in mesenchymalstem cell line C3H10T1/2. FIG. 1a shows the RT-PCR analyses ofBMP2-dependent expression of FGF- and PTH/PTHrP-receptors in mesenchymalstem cell line C3H10T1/2 in the presence or absence of recombinantlyexpressed BMP2. FIG. 1b shows the effect of cyclohexamide pretreatmentof C3H10T1/2 cells. Cycloheximide pre-treatment of C3H10T1/2 cells doesnot prevent BMP-induction of the FGFR3 gene. Cells were mock-treated(control) or were treated with BMP2 (50 ng/ml). FIG. 1c demonstrateswestern immunoblotting for the detection of BMP2-dependent FGFR3 andFGFR2 expression in cellular extracts of C3H10T1/2 lines. FIG. 1d leftpanel: western immunoblotting to demonstrate the forced expression ofFGFR3 in mesenchymal progenitors C3H10T1/2; right panel: the recombinantexpression of FGFR3 in C3H10T1/2 leads to enhanced levels of activatedMAP-kinases pERK-1 and pERK-2 during cultivation. Cell lysates wereprepared 0 (=confluence) and 4 days post-confluence. FIG. 1edemonstrates that the forced expression of FGFR3 in parental C3H10T1/2cells is sufficient for the induction of the chondrogenic lineage.

[0023]FIG. 2. The T-box transcription factor Brachyury mediateschondrogenic differentiation in MSCs in vitro and ectopically in vivo.FIG. 2a upper panel: schematic representation of Brachyury according toKispert et al., 1995 (1995). FIG. 2a lower panel: western immunoblottingof recombinant HA-tagged Brachyury (aa 1-436) in cellular extracts ofC3H10T1/2 (C3H10T1/2-Brachyury) with HA-antibody SC-805 (Santa Cruz)Brachyury has been constitutively expressed under the control of themurine PGK-promoter. Expression of Brachyury is indicated (triangle).Molecular weight marker (M) shown is ovalbumin (43 kDa). FIG. 2b showsthe histological characterization of C3H10T1/2-Brachyury cells inculture. Upper panel: at day 4 post-confluency cells developalkaline-phosphates positive osteoblast-like cells. Lower right panel:Alcian Blue histology of C3H10T1/2 cells stably expressing Brachyuryindicative for secreted proteoglycans and efficient differentiation intothe chondrogenic lineage. Lower left panel:Collagen-immunohistochemistry of C3H10T1/2-Brachyury cells in culture 7days post-confluency. FIG. 2c shows the RT-PCR analysis of theexpression of chondrogenic and osteogenic marker genes in C3H10T1/2cells recombinantlly-expressing Brachyury. FIG. 2d shows the forcedexpression of the T-box factor Brachyury in C3H10T1/2 cells which leadsto differentiation into chondrocytes and cartilage development at murineectopic sites after intramuscular transplantation.

[0024]FIG. 3. Dominant-negative Brachyury (dnBrachyury; T-box domain)blocks BMP2-mediated chondrogenic development in C3H10T1/2 MSCs in vitroand ectopically in vivo. FIG. 3a shows that Brachyury's T-box domaininterferes with the transcriptional activity of full-length Brachyury.FIG. 3b shows expression of dnBrachyury (T-box domain) in C3H10T1/2-BMP2during cultivation (day 0; cellular confluence) The T-box domain (aa1-229) has been subcloned and HA-tagged in expression vector pMT7T3 andconstitutively expressed in C3H10T1/2-BMP2 cells. The recombinantllyexpressed T-box domain (dnBrachyury) is indicated (triangle). FIG. 3cdemonstrates RT-PCR experiments with osteo/chondrogenic marker genesshow that T-box domain (dnBrachyury) expression in C3H10T1/2-BMP2 cellsinterferes with the BMP2 dependent of FGFR2 but not FGFR3 expression.FIG. 3d shows that dnBrachyury (T-box) interferes with BMP2-mediatedFGFR2 expression as analyzed by western immnunoblotting with antiFGFR3and antiFGFR2 antibodies as described FIG. 1. FIG. 3e shows the forcedexpression of the dominant-negative acting T-box domain inC3H10T1/2-BMP2 cells interferes with BMP-2 mediated osteo-/chondrogenicdevelopment.

[0025]FIG. 4. Dominant-negative FGFR3 (dnFGFR3) interferes withosteo-/chondrogenic development, with FGFR2- and withBrachyury-expression in C3H10T1/2-BMP2. FIG. 4a shows that forcedexpression of dnFGFR3 in C3H10T1/2-BMP2 cells interferes with BMP-2mediated development of alkaline phosphatase positive and Alcian Bluepositive chondrocyte-like cells, respectively. FIG. 4b shows thatdnFGFR3 interferes with BMP2-dependent FGFR2 and Brachyury but not withFGFR3 expression in C3H10T1/2-BMP2 cells.

[0026]FIG. 5. FGFR3 and Brachyury are involved in an auto regulatoryloop. FIG. 5a shows RT-PCR analyses of FGFR3 and Brachyury mRNA levelsin mesenchymal progenitors C3H10T1/2 expressing recombinant FGFR3(C3H10T1/2-FGFR3) or Brachyury (C3H10T1/2-Brachyury). FIG. 5b shows thatSmad1-signaling is not sufficient for Brachyury and FGFR3 but forosteocalcin expression. RT-PCR analyses of FGFR3 and Brachyury mRNAlevels in mesenchymal progenitors C3H10T1/2 expressing the biologicallyactive Smad1-MH2 domain (C3H10T1/2-Smad1-MH2).

[0027]FIG. 6. Brachyury is expressed at skeletal sites during latemurine embryonic development (18.5 dpc). Comparative expression analysisof murine Brachyury (Bra), Collagen 1a1 (Co1 1a1) and Collagen 2a1 (Co12a1) in embryonic development 18.5 dpc. a, Intervertebral discsdevelopment. Consecutive sagittal (a-g) and transversal (h-j) sectionsof 18.5 dpc mouse embryos were hybridized with riboprobes as indicated.Expression of Brachyury is enhanced in the nucleus pulposus (a, d), Co11a1 in the outer annulus (arrowheads in b, e), and Co1 2a1 in thecartilage primordium of the vertebrae (c,f). No signals are obtainedusing RNase pre-incubated sections (g). With transversal sections at thelevel of the upper lumbar vertebra expression of Brachyury is inaddition detectable in distinct cells of the neural arch (h) whereasCo1a1 is expressed in the outer annulus (i) and Co1 2a1 in the cartilageprimordium (j). b, Limb bud development. Consecutive transversalsections of a 18.5 dpc mouse hind limb at the level of the metatarsalshybridized with riboprobes as indicated. Expression of Brachyury isevident in distinct chondrogenic cells of the forming metatarsal bones(a), better visible with higher magnification (b,c). In contrast Co1 1a1is expressed in the outer periosteal layer (d-f) and Co1 2a1 expressionis enhanced in differentiating chondrocytes (g-i). As it was evident forthe intervertebral disc formation expression of Brachyury is onlyevident in chondrocyte-like cells that do not express Co1 2a1. ch,chondrocytes; cp, cartilage primordium; mta, metatarsal; np, nucleuspulposus; oa, outer annulas; pl, periosteal layer; sk, skin bar, 100 □m

[0028]FIG. 7. Model of BMP2-dependent osteo-/chondrogenic development inmesenchymal stem cells.

[0029]FIG. 8. Human adult MSCs (3×106) were transfected with 30 ug ofBrachyury plasmid. 24 hours post transfection, RNA was isolated andRT-PCR was performed using specific primers to the Brachyury cDNA. 20 ulof the PCR reaction sample were loaded and electrophoresed in 2% Agarosegel. The gel image demonstrated positive Brachyury expression (Positivecontrol—Brachyury plasmid).

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0030] In one embodiment, the invention relates to methods of enhancingrepair of a cartilage and/or inducing formation of a cartilage byadministering a cell, which expresses a factor of the T-box family,which includes inter-alia the brachyury. In another embodiment, theinvention relates to an engineered cell, which is transfected with avector comprising a nucleic acid sequence encoding a factor of the T-boxfamily, thereby expressing a factor of the T-box family. In anotherembodiment, the invention relates to compositions comprising a vector,which comprises a nucleic acid sequence encoding a factor of the T-boxfamily and in another embodiment the composition-comprising cell thatexpresses a factor of the T-box family, which includes the brachyury.

[0031] The term “cartilage” refers hereinabove to a specialized type ofdense connective tissue consisting of cells embedded in a matrix. Thereare several kinds of cartilage. Translucent cartilage having ahomogeneous matrix containing collagenous fibers is found in articularcartilage, in costal cartilages, in the septum of the nose, in larynxand trachea. Articular cartilage is hyaline cartilage covering thearticular surfaces of bones. Costal cartilage connects the true ribs andthe sternum. Fibrous cartilage contains collagen fibers. Yellowcartilage is a network of elastic fibers holding cartilage cells whichis primarily found in the epiglottis, the external ear, and the auditorytube. Cartilage is tissue made up of extracellular matrix primarilycomprised of the organic compounds collagen, hyaluronic acid (aproteoglycan), and chondrocyte cells, which are responsible forcartilage production. Collagen, hyaluronic acid and water entrappedwithin these organic matrix elements yield the unique elastic propertiesand strength of cartilage.

[0032] As used herein, “hyaline cartilage” refers to the connectivetissue covering the joint surface. By way of example only, hyalinecartilage includes, but is not limited to, articular cartilage, costalcartilage, and nose cartilage.

[0033] As used herein, the term “enhancing cartilage repair” refers tohealing and for regeneration of cartilage injuries, tears, deformitiesor defects, and prophylactic use in preventing damage to cartilaginoustissue.

[0034] As used herein, the term “inducing formation” refers to the usein cartilage renewal or regeneration so as to ameliorate conditions ofcartilage, degeneration, depletion or damage such as might be caused byaging, genetic or infectious disease, accident or any other cause, inhumans, livestock, domestic animals or any other animal species. Inanother embodiment the formation of a cartilage is required forcartilage development in livestock, domestic animals or any other animalspecies in order to achieve increased growth for commercial or any otherpurpose. In another embodiment the formation of a cartilage is requiredin plastic surgeries, such as without being limited facialreconstruction in order to obtain a stabilized shape.

[0035] In one embodiment there is provided a recombinant vectorcomprising a nucleic acid sequence encoding a factor of the T-boxfamily.

[0036] The term “T-box family” defined as a family of transcriptionfactors that share the T-box, a 200 amino acid DNA-binding domain(T-box, aa 1-229). The T-box family has been identified in bothvertebrates and in vertebrates and plays a key role in embryonicdevelopment. The T-box family further includes variant and fragments ofthe T-box family transcription factors.

[0037] The T-Box gene family can be said to consist of several genericentities: T, Tbr-1, Tbx1-9, 11, 12, 17 and T2 and many species has beenshown to contain orthologs. Several mouse T-Box genes have beenreported; mu-T, mu-Tbr1 (identified in a subtractive hybridizationscreen for genes specifically involved in regulating forebraindevelopment (Bulfone et al. (1995) Neuron 15:63-78), mu-Tbx1-6, mm-Tbx13(Wattler et al., Genomics 48:24-33), and mm-Tbx14 (Wattler et al. (1998)Genomics 48:24-33, 1998). There are four Xenopus genes (Xbra, x-eomes,x-ET and x-VegT (Zhang et al. (1996) Development 122:4119-4129; Smith etal. (1995) Semin Dev Biol 6:405-410; Lustig et al. (1996) Development122:4001-4012; Stennard et al. (1996) Development 122:4179-4188; Horb etal. (1997) Development 124:1689-1698; Ryan et al. (1996) Cell87:989-1000). Human orthologs for six of eight mouse genes have beenidentified. Hu-T (Edwards et al. (1996) Genome Res 6:226-233; Morrisonet al. (1996) Hum Mol Genet 5:669-674) and hu-TBRI (Bulfone et al.(1995) Neuron 15:63-78) were found by homology with the mouse orthologs.Hu-TBX2 was isolated independently by two groups from embryonic kidneycDNA libraries (Campbell et al. (1995) Genomics 28:255-260; Law et al.(1995) Mamm Genome 6:267-277). Hu-TBX1, hu-TBX3, and hu-TBX5 were foundduring investigations aimed at uncovering the genetic basis of humandevelopmental dysmorphic syndromes and were recognized as orthologs ofthe mouse genes by sequence homology (Li et al. (1997) Nat Genet15:21-29; Basson et al. (1997) Nat Genet 15:30-35; Chieffo et al. (1997)Genome 43:267-277).

[0038] There is currently only a handful of known mutations in T-Boxgenes. Spontaneous mutations in hu-TBX3 (Bamshad et al. (1997) Nat Genet16:31 1-315) and hu-TBX5 (Li et al. (1997) Nat Genet 15:21-29; Basson etal. (1997) Nat Genet 15:30-35) have been reported. These mutations atT-Box genes play a role in several human autosomal, dominantdevelopmental syndromes: Ulnar-Mammary syndrome and Holt-Oram syndrome.Ulnar-Mammary syndrome is characterized by limb defects, abnormalitiesof apocrine glands such as the absence of breasts, axillary hair andperspiration, dental abnormalities such as ectopic, hypoplastic andabsent canine teeth, and genital abnormalities such as micropenis, shawlscrotum and imperforate hymen. Holt-Oram syndrome is characterized bycardiac septal defects and preaxial radial ray abnormalities of theforelimbs (Li et al. (1997) Nat Genet 15:21-29; Basson et al. (1997) NatGenet 15:30-35; Barnshad et al. (1997) Nat Genet 16:311-315). Mutationsin the 5′ end of TBX5 lead to substantial cardiovascular malformationsand relatively mild skeletal defects while mutations in the 3′ end ofthe gene cause severe skeletal malformation and have less effect oncardiac development (McCarthy, M (1998) Lancet 351(9115):1564; Basson,C. T. et al (1997) Nature Genetics 15:30-35). A better understanding ofthe role which T-Box transcription factors play in embryogenesis,organogenesis and organ regeneration has been recently recognized. T-Boxrelated genes have been found in many species, making up a large groupof T-Box transcription factors which are highly conserved in theirDNA-binding capacity but may be highly divergent in the non-DNA-bindingregions. There are common features which define the family, as well asspecific differences that define individual members. Phylogeneticanalysis suggests that the genome of most animal species will have atleast five T-Box genes (related to mu-Tbx2, mu-Tbx, mu-Tbx1, mu-T, andmu-Tbr1). There are at least 16 distinct members in 11 different animalgroups that have been reported and human orthologs of six of the eightmouse genes have already been identified.

[0039] In another embodiment, the vector comprising a nucleic acid whichencodes for Brachyury, or T, which refers hereinabove to the founderfactor of the T-box family.

[0040] The immediate BMP2-dependent upregulation of FGFR3 in MSCs(C3H10T1/2) and the inherent capacity of this receptor to initiatechondrogenic development in these cells prompted a screen forFGFR3-regulated transcription factors. The chondrogenic potential ofBrachyury after recombinant expression in wild-type C3H10T1/2 cells hasbeen shown, by the use of a subtractive screening method, exemplified inExample 1 that, among the transcription factors tested, the T-boxtranscription factor Brachyury was upregulated in FGFR3-expressingC3H10T1/2 cells (see also FIG. 5a).

[0041] As used herein, the term “nucleic acid” refers to polynucleotidesor to ologonucleotides such as deoxyribonucleic acid (DNA), and, whereappropriate, ribonucleic acid (RNA) or mimetics thereof. The term shouldalso be understood to include, as equivalents, analogs of either RNA orDNA made from nucleotide analogs, and, as applicable to the embodimentbeing described, single (sense or antisense) and double-strandedpolynucleotides. This term includes oligonucleotides composed ofnaturally occurring nucleobases, sugars and covalent internucleoside(backbone) linkages as well as oligonucleotides having non-naturallyoccurring portions which function similarly. Such modified orsubstituted oligonucleotides are often preferred over native formsbecause of desirable properties such as, for example, enhanced cellularuptake, enhanced affinity for nucleic acid target and increasedstability in the presence of nucleases.

[0042] The terms “protein”, “polypeptide” and “peptide” are usedinterchangeably herein when referring to a gene product.

[0043] The vector molecule can be any molecule capable of beingdelivered and maintained within the target cell or tissue such that thegene encoding the product of interest can be stably expressed. Thevector molecule preferably utilized in the present invention is either aviral or retroviral vector molecule or a plasmid DNA non-viral molecule.This method preferably includes introducing the gene encoding theproduct into the cell of the mammalian connective tissue for atherapeutic or prophylactic use. Unlike previous pharmacologicalefforts, the methods of the present invention employ gene therapy toaddress the chronic debilitating effects of joint pathologies. The viralvectors used in the methods of the present invention can be selectedform the group consisting of (a) a retroviral vector, such as MFG orpLJ; (b) an adeno-associated virus; (c) an adenovirus; and (d) a herpesvirus, including but not limited to herpes simplex 1 or herpes simples 2or (e) lentivirus. Alternatively, a non-viral vector, such as a DNAplasmid vector, can be used. Any DNA plasmid vector known to one ofordinary skill in the art capable of stable maintenance within thetargeted cell or tissue upon delivery, regardless of the method ofdelivery utilized is within the scope of the present invention.Non-viral means for introducing the gene encoding for the product intothe target cell are also within the scope of the present invention. Suchnon-viral means can be selected from the group consisting of (a) atleast one liposome, (b) Ca3 (PO4) 2, (c) electroporation, (d)DEAE-dextran, and (e) injection of naked DNA.

[0044] As will be appreciated by one skilled in the art, a fragment orderivative of a nucleic acid sequence or gene that encodes for a proteinor peptide can still function in the same manner as the entire, wildtype gene or sequence. Likewise, forms of nucleic acid sequences canhave variations as compared with the wild type sequence, while thesequence still encodes a protein or peptide, or fragments thereof, thatretain their wild type function despite these variations. Proteins,protein fragments, peptides, or derivatives also can experiencedeviations from the wild type from which still functioning in the samemanner as the wild type form. Similarly, derivatives of the genes andproducts of interest used in the present invention will have the samebiological effect on the host as the non-derivatized forms. Examples ofsuch derivatives include but are not limited to dimerized oroligomerized forms of the genes or proteins, as wells as the genes orproteins modified by the addition of an immunoglobulin (Ig) group.Biologically active derivatives and fragments of the genes, DNAsequences, peptides and proteins of the present invention are thereforealso within the scope of this invention. In addition, any nucleic acid,which is cis acting and integrated upstream to an endogenous factor ofthe T-box family nucleic acid sequence, is relevant to the presentinvention.

[0045] The term “cis-acting” is used to describe a genetic region thatserves as an attachment site for DNA-binding proteins (e.g. enhancers,operators and promoters) thereby affecting the activity of genes on thesame chromosome.

[0046] It was shown that Brachyury expression is upregulated by certainfactor such as BMP2 and/or FGF3 (see FIGS. 4 and 5). Thus, in anotherembodiment, the vector of the invention further comprises a nucleicacid, which encodes to a protein, which activated the BMP signalingpathway. In another embodiment, the protein, which activated the BMPsignaling pathway, is a member of the BMP family. In another embodimentthe BMP is a BMP2. In another embodiment the vector further comprising anucleic acid for fibroblast growth factor namely FGF-3.

[0047] The term “protein which activates BMP mediated signaling pathway”is defined hereinabove as a protein that can activate the BMP receptors,or the signaling cascade down stream of the receptor to elicit BMPspecific cellular response. Examples, without being limited are membersof the BMP family, such as the BMP proteins BMP-1, BMP-2, BMP-3, BMP-4,BMP-5, BMP-6 and BMP-7, disclosed for instance in U.S. Pat. Nos.5,108,922; 5,013,649; 5,116,738; 5,106,748; 5,187,076, and 5,141,905;BMP-8, disclosed in PCT publication W091/18098; BMP-9, disclosed in PCTpublication W093/00432; and BMP-10 or BMP-11, disclosed in co-pendingpatent applications, Ser. No. 08/061,695 presently abandoned, acontinuation-in-part of which has issued as U.S. Pat. No. 5,637,480, and08/061,464 presently abandoned, a continuation-in-part of which hasissued as U.S. Pat. No. 5,639,638 filed on May 12, 1993.

[0048] In another embodiment the vector may include also nucleic acidsencoding other therapeutically useful agents including MP52, epidermalgrowth factor (EGF), fibroblast growth factor (FGF), platelet derivedgrowth factor (PDGF), transforming growth factors (TGF-αand TGF-β), andfibroblast growth factor-4 (FGF-4), parathyroid hormone (PTH), leukemiainhibitory factor (LIF/HILDA/DIA), insulin-like growth factors (IGF-Iand IGF-II). In another embodiment, the vector comprises nucleic acidencoding an anti-inflammatory agent such as IL1 receptor antagonist, orIL4 or IL10 agonist.

[0049] In another embodiment the cell of the invention further expressestherapeutically useful agents including MP52, epidermal growth factor(EGF), fibroblast growth factor (FGF), platelet derived growth factor(PDGF), transforming growth factors (TGF-αand TGF-β), and fibroblastgrowth factor-4 (FGF-4), parathyroid hormone (PTH), leukemia inhibitoryfactor (LIF/HILDA/DIA), insulin-like growth factors (IGF-I and IGF-II).In another embodiment, the vector comprises nucleic acid encoding ananti-inflammatory agent such as IL1 receptor antagonist, or IL4 or IL10agonist.

[0050] In another embodiment, there is provided an engineered cell,which expresses at least one factor of the T-box family.

[0051] The term “engineered cell” is defined hereinabove to a cell or toa tissue, which had been genetically modified and is expressing a factorof the T-box family or in another embodiment, increased amounts of thefactor of the T-box family or in another embodiment express Brachury.The term “increased amount of the factor or the at least one factor ofthe T-box family refers hereinabove to at least 10 times more thannormal.

[0052] In one embodiment, the cell of the invention is a mammalian cell.In another embodiment, it is a mesenchymal stem cell, in anotherembodiment it is a progenitor cell, in another embodiment it is a cellderived from a cartilage. In another embodiment the cell can be derivedfrom a fibroblast cell line, a mesenchymal cell line, a chondrocyte cellline, an osteoblast cell line, or an osteocyte cell line. The fibroblastcell line may be a human foreskin fibroblast cell line or NIH 3T3 cellline. In another embodiment the cell of the invention is a synovial cellor a synoviocyte. Synoviocytes are found in joint spaces adjacent tocartilage have an important role in cartilage metabolism. Synoviocytesproduce metallo-proteinases, such as collagenases that are capable ofbreaking-down cartilage

[0053] Stem cells are defined as cells which are not terminallydifferentiated, which can divide without limit, and divides to yieldcells that are either stem cells or which irreversibly differentiate toyield a new type of cell. Those stem cells which give rise to a singletype of cell are call unipotent cells; those which give rise to manycell types are called pluripotent cells. Chondro/osteoprogenlitor cells,which are bipotent with the ability to differentiate into cartilage orbone, were isolated from bone marrow (for example, as described by Owen,J. Cell Sci. Suppl. 10, 63-76 (1988) and in U.S. Pat. No. 5,226,914 toCaplan, et al.).

[0054] It is important to note that mesenchymal stem cells andprogenitors can be isolated from different source tissues, skin, bonemarrow, muscle, and liver. In addition any cell type with stem cellproperties or demonstrating differentiation plasticity for examplewithout limitation, SP cells from the source of bone marrow, muscle,spleen or any other tissue.

[0055] Chondrogenic cells useful in the practice of the invention may beisolated from essentially any tissue containing chondrogenic cells. Asused herein, the term “chondrogenic cell” is understood to mean any cellwhich, when exposed to appropriate stimuli, may differentiate into acell capable of producing and secreting components characteristic ofcartilage tissue. The chondrogenic cells may be isolated directly frompreexisting cartilage tissue, for example, hyaline cartilage, elasticcartilage, or fibrocartilage. Specifically, chondrogenic cells may beisolated from articular cartilage (from either weight-bearing ornon-weight-bearing joints), costal cartilage, nasal cartilage, auricularcartilage, 30 tracheal cartilage, epiglottic cartilage, thyroidcartilage, arytenoid cartilage and cricoid cartilage. The cell from thecartilage can be derived from another animal, or another subject or inanother embodiment; the cell of the cartilage or the bone can be derivedfrom the subject in need.

[0056] In another embodiment, the cell further expresses at least oneprotein, which activates BMP mediated signaling pathway or a FGFprotein.

[0057] The expression of the at least one factor of the T-box family incombination with either and BMP or FGF or both in the cell can be due tothe presence of two or more different vectors (trans vectors) or due tothe expression of one vector which comprises two or more differentnucleic acid sequences, which encode for the at least one member of theT-box family, the FGF and for at least one protein which activates theBMP mediated signaling pathway. It was shown that Brachyury'sDNA-binding domain without the associated regulatory domains (aa230-436) should dominant-negatively (dn) interfere with endogenousBrachyury-mediated events in C3H10T1/2-BMP2 cells see FIG. 3b andexample 3.

[0058] In another embodiment the invention provides complex tissueengineering. This term refers to engineering a cell with differentnucleic acid sequences, wherein each sequence encodes to a specificpathway of differentiation. As such, the cell of the invention can beengineered to differentiate to an osteoblast as well as to achondrocyte.

[0059] In another embodiment there is provided a composition comprisingrecombinant vector comprising a nucleic acid sequence encoding the afactor of the T-box family and a pharmaceutically acceptable carrier. Itshould be noted that the term “a nucleic acid sequence encoding the afactor of the T-box family” refers hereinabove to “at least one nucleicacid sequence encoding the at least one factor of the T-box family”.Similarly the term “a cell” refers to “at least one cell”.

[0060] In another embodiment, there is provided a composition comprisingat least one engineered cell, wherein said engineered cell expressesleast one factor of the T-box family at least one protein and apharmaceutically acceptable carrier.

[0061] In another embodiment the composition can be a pharmaceuticalcomposition.

[0062] Compositions of the invention may further comprise additionalproteins, such as additional factors. These compositions may be used toinduce the formation or repair of cartilage tissue.

[0063] The compositions of the invention may comprise, also BMP-12 orVL-1 (BMP-13), other therapeutically useful agents including MP52,epidermal growth factor (EGF), fibroblast growth factor (FGF), plateletderived growth factor (PDGF), transforming growth factors (TGF-□andTGF-□), and fibroblast growth factor-4 (FGF-4), parathyroid hormone(PTH), leukemia inihibitory factor (LIF/HILDA/DIA), insulin-like growthfactors (IGF-I and IGF-II). Portions of these agents may also be used incompositions of the present invention. N another embodiment thecomposition comprises anti-inflammatory agents such as IL1 receptorantagonists, or IL4 or IL10 agonists.

[0064] In another embodiment, there is provided an implant device fortransplantation in a subject in need comprising an engineered cell whichexpresses a factor of the T-box family and a pharmaceutically acceptablecarrier. Cartilage implants are often used in reconstructive or plasticsurgery such as rhinoplasty.

[0065] The preparation and formulation of suchpharmaceutically/physiologically acceptable protein compositions, havingdue regard to pH, isotonicity, stability and the like, is within theskill of the art. The therapeutic compositions are also presentlyvaluable for veterinary applications due to the lack of speciesspecificity in factor of the T-box family due to high homology betweenspecies.

[0066] Particularly domestic animals and thoroughbred horses in additionto humans are desired patients for such treatment with the compositionsof the present invention.

[0067] The therapeutic method includes administering the compositiontopically, systemically, or locally as an injectable and/or implant ordevice. When administered, the therapeutic composition for use in thisinvention is, of course, in a pyrogen-free, physiologically acceptableform. Further, the composition may desirably be encapsulated or injectedin a viscous form for delivery to the site of tissue damage.Therapeutically useful agents other than the proteins, which may alsooptionally be included in the composition, as described above, mayalternatively or additionally, be administered simultaneously orsequentially with the composition in the methods of the invention. Inaddition, the compositions of the present invention may be used inconjunction with presently available treatments for cartilage injurysuch as cartilage allograft or autograft, in order to enhance oraccelerate the healing potential of the or graft. For example, the,allograft or autograft may be soaked in the compositions of the presentinvention prior to implantation. It may also be possible to incorporatethe protein or composition of the invention onto suture materials, forexample, by freeze-drying.

[0068] The compositions may include an appropriate matrix and/orsequestering agent as a carrier. For instance, the matrix may supportthe composition or provide a surface for cartilage-like tissueformation. The matrix may provide slow release of the protein and/or theappropriate environment for presentation thereof. The sequestering agentmay be a substance, which aids in case of administration throughinjection or other means, or may slow the migration of protein from thesite of application.

[0069] The choice of a carrier material is based on biocompatibility,biodegradability, mechanical properties, cosmetic appearance andinterface properties. The particular application of the compositionswill define the appropriate formulation. Potential matrices for thecompositions may be biodegradable and chemically defined. Furthermatrices are comprised of pure proteins or extracellular matrixcomponents. Other potential matrices are non-biodegradable andchemically defined. Preferred matrices include collagen-based materials,including sponges, such as Helistat.RTM. (Integra LifeSciences,Plainsboro, N.J.), or collagen in an injectable form, as well assequestering agents, which may be biodegradable, for example hyalouronicacid derived. Biodegradable materials, such as cellulose films, orsurgical meshes, may also serve as matrices. Such materials could besutured into an injury site, or wrapped around the tendon/ligament.

[0070] Another preferred class of carrier are polymeric matrices,including polymers of poly (lactic acid), poly(glycolic acid) andcopolymers of lactic acid and glycolic acid. These matrices may be inthe form of a sponge, or in the form of porous particles, and may alsoinclude a sequestering agent. Suitable polymer matrices are described,for example, in W093/00050, the disclosure of which is incorporatedherein by reference.

[0071] Preferred families of sequestering agents include blood, fibrinclot and/or cellulosic materials such as alkylcelluloses (includinghydroxyalkylcelluloses), including methylcellulose, ethylcellulose,hydroxyetlhylcellulose, hydroxypropylcellulose,hydroxypropyl-methylcellulose, and carboxymethylcellulose, the mostpreferred being cationic salts of carboxymethylcellulose (CMC). Otherpreferred sequestering agents include hyaluronic acid, sodium alginate,poly (ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer andpoly(vinyl alcohol). Tie amount of sequestering agent useful herein is0.5-20 wt %, preferably 1-10 wt % based on total formulation weight,which represents the amount necessary to prevent desorption of theprotein from the polymer matrix and to provide appropriate handling ofthe composition, yet not so much that the progenitor cells are preventedfrom infiltrating the matrix, thereby providing the protein theopportunity to assist the activity of the progenitor cells.

[0072] Additional optional components useful in the practice of thesubject application include, e.g. cryogenic protectors such as mannitol,sucrose, lactose, glucose, or glycine (to protect the protein fromdegradation during lyophilization), antimicrobial preservatives such asmethyl and propyl parabens and benzyl alcohol; antioxidants such asEDTA, citrate and BHT (butylated hydroxytoluene); and surfactants sifdchas poly(sorbates) and poly(oxyethylenes); etc.

[0073] As described above, the compositions and the devices of theinvention may be employed in methods for enhancing cartilage repair orfor inducing cartilage formation. These methods, according to theinvention, entail administering to a patient needing such tissue repair;a cell expresses at least one factor of the T-box family or in anotherembodiment a composition comprising an effective amount of vectorcomprising a nucleic acid encoding a factor of the T-box family.

[0074] In another embodiment, as described before, the composition orthe cell may comprise also a vector comprising a nucleic acid encodingFGF and/or a factor of the BMP family.

[0075] Preferably the DNA molecule or protein may be injected directlyinto cartilage tissue such as without limitation nasal cartilage,articular cartilage etc. Therefore, the compounds of the invention maybe utilized as a therapeutic agent in regard to treatment of cartilageor bone damage caused by disease or aging or by physical stress such asoccurs through injury or repetitive strain, e.g. “tennis elbow” andsimilar complaints. The therapeutic agent of the invention may also beutilized as part of a suitable drug delivery system to a particulartissue that may be targeted.

[0076] Other therapeutic applications for the compounds of the inventionmay include the following: 1. Use in cartilage and/or bone renewal,regeneration or repair so as to ameliorate conditions of cartilageand/or bone breakage, degeneration, depletion or damage such as might becaused by aging, genetic or infectious disease, wear and tear, physicalstress (for example, in athletes or manual laborers), accident or anyother cause, in humans, livestock, domestic animals or any other animalspecies; 2. Stimulation of skeletal development in livestock, domesticanimals or any other animal species in order to achieve increased growthfor commercial or any other purpose; 3. Treatment of neoplasia orhyperplasia of bone or cartilage, in humans, livestock, domestic animalsor any other animal, species; 4. Suppression of growth of skeletalcomponents in livestock, domestic animals or any other animal species inorder to achieve decreased growth for commercial or any other purposese.g. by the use of anti sense molecules to the factor of the T-boxfamily; and 5. Alteration of the quality or quantity of cartilage and/orbone for any other purpose in any animal species including humans.

[0077] Thus, according to clauses 4 and 5 the invention can be servealso for suppressing cartilage formation, by the use of an anitagonistto Brachyury or to other factors of the T-box family. The antagonisticeffect of dominant negative Brachyury is exemplified in Example 3. Theterm “antagonist” refers to a molecule which, when bound to the epitope,decreases the amount or the duration of the effect of the biological orimmunological activity of epitope. Antagonists may include proteins,nucleic acids, carbohydrates, antibodies, anti sense or any othermolecules, which decrease the effect of Brachyury or to other factors ofthe T-box family on cartilage formation. Such a treatment of suppressionis relevant in the treatment of malignancy of the cartilage, for examplewithout limitation in chondronia and chondrasarcoma. In anotherembodiment the antagonist is a dominant negative factor of the T-boxfamily. In another embodiment the antagonist is a dominant negativeBrachyury.

[0078] The term “dominant negative Brachyury refers hereinabove toBrachyury DNA binding domain (T-box, aa 1-229) without the associatedregulatory domains (aa 230-436).

[0079] In another embodiment the invention provides a method of inducingchondrocyte differentiation comprising the step of administering of arecombinant vector, which comprises a nucleic acid encoding a factor ofthe T-box family, thereby inducing chondlocyte formation. Example 2 andFIG. 2 clearly demonstrates that forced expression of the T-box factorBrachyury leads to chondrogenic development in C3H10T1/2 mesenchymalstem cells. Please note that C3H10T1/2 mesenclhymal stem cells line.C3H10T1/2 stem cell line resembles human MSCs by many features includingdifferentiation multi potentiality, high proliferation capacity andsimilar response to growth factors and cytokines such as BMPs.

[0080] Example 3 further strengthen the relation between Brachyury andchondrogenic development, by demonstrating that dominant negativeBrachyury interferes with BMP2 dependent chondrogenic development inmesenchymal cells.

[0081] In another embodiment the invention relates to a method for theproduction of transplantable cartilage matrix, the method comprising thesteps of: obtaining a cell; transfecting said cell with a recombinantvector comprising a nucleic acid sequence encoding a factor of the T-boxfamily, so as to obtain an engineered cell which expresses a factor ofthe T-box family; and culturing said cell with the cell-associatedmatrix for a time effective for allowing formation of a transplantablecartilage matrix. The above method will enable the production of acartilage matrix, which will be transplanted to a subject in need whenrequired.

[0082] In another embodiment, there is provided a method of treating asubject by ex-vivo implantation of at least one cell comprising thefollowing steps: obtaining at least one cell from the subject;transfecting the cell with a nucleic acid which encodes at least onefactor of the T-box family, so as to obtain ail cell which express atleast one factor of the T-box family activated cell; and administeringsaid activated cell to the subject.

[0083] Optionally, the enriched stem cells are then expanded ex vivo byculturing them in the presence of agents that stimulate proliferation ofstem cells. The culturing step can be for example in a bioreactor, whichenables thee dimensional growth of the cells. The enriched andoptionally expanded stem cells are then infected with a vector, thatexpresses the at least one factor of the T-box family gene. Optionally,the vector may also carry an expressed selectable marker, in which casesuccessfully transduced cells may be selected for the presence of theselectable marker. The transduced and optionally selected stem cells arethen returned to the patient defective connective tissue and allowed toengraft themselves into the bone marrow.

[0084] One ex vivo method of enhancing repair and/or inducing formationdisclosed throughout this specification comprises initially generating arecombinant viral or plasmid vector which contains a DNA sequenceencoding a protein or biologically active fragment thereof. Thisrecombinant vector is then used to infect or transfect a population ofin vitro cultured connective tissue cells, resulting in a population ofconnective cells containing the vector. These connective tissue cellsare then transplanted to a target joint space of a mammalian host,effecting subsequent expression of the protein or protein fragmentwithin the joint space. Expression of this DNA sequence of interest isuseful in substantially reducing at least one deleterious jointpathology associated with a connective tissue disorder.

[0085] It will be understood by the artisan of ordinary skill, that tiesource of cells for treating a human patient is the patient's ownconnective tissue cells, such as autologous fibroblast cells. In anotherembodiment the source of cells can be allogenic cells, which weretreated so as to reduce immune response.

[0086] As used herein, a “promoter” can be any sequence of DNA that isactive, and controls transcription in a eucaryotic cell. The promotermay be active in either or both eucaryotic and procaryotic cells. Inanother embodiment, the promoter is active in mammalian cells. Thepromoter may be constitutively expressed or inducible. In anotherembodiment, the promoter is inducible. In another embodiment, thepromoter is inducible by an external stimulus. In another embodiment,the promoter is inducible by hormones or metals. Still more in anotherembodiment, the promoter is inducible by heavy metals. In anotherembodiment, the promoter is a metallothionein gene promoter. In anotherembodiment the promoter is inducible by antibiotics such astetracycline. In another embodiment the promoter is inducible by atissue specific promoter. Likewise, “enhancer elements”, which alsocontrol transcription, can be inserted into the DNA vector construct,and used with the construct of the present invention to enhance theexpression of tie gene of interest.

[0087] In another embodiment there provided ex vivo and in vivotechniques for delivery of a DNA sequence of interest to tie connectivetissue cells of the mammalian host. The ex vivo technique involvesculture of target connective tissue cells, in vitro transfection of theDNA sequence, DNA vector or other delivery vehicle of interest into theconnective tissue cells, followed by transplantation of the modifiedconnective tissue cells to the target joint of the mammalian host, so asto effect in vivo expression of the gene product of interest.

[0088] Alternatively, an allograft, (e.g., cartilage grown in vitro fromcartilage tissue removed from the patient) may be implanted by attachinga periosteum membrane (harvested, e.g., from the patient's tibia), tothe bone surface and injecting the allograft beneath the membrane.

[0089] As used herein, the term “transfection” means the introduction ofa nucleic acid, e.g., an expression vector, into a recipient cell bynucleic acid-mediated gene transfer.

[0090] Alternatively, the gene encoding the product of interest can beassociated with liposomes and injected directly into the host, such asin the area of the joint, where the liposomes fuse with target cells,resulting in an in vivo gene transfer to the connective tissue. Inanother embodiment, the gene encoding the product of interest isintroduced into the area of the joint as naked DNA. The naked DNA entersthe target cells, resulting in an in vivo gene transfer to the cells.

[0091] The dosage of the treatment, which is the amount of the cellswhich express the at least one factor of the T-box family or in anotherembodiment the amount of the composition or the device which contain thevector comprising the nucleic acid encoding the same in the in vivo andin the ex-vivo treatment the dosage regimen will be determined by theattending physician considering various factors which modify the actionof the composition, e.g., amount of bone or cartilage tissue desired tobe formed, the site of the bone or cartilage damage, the condition ofthe damaged cartilage or bone, the size of a wound, type of damagedtissue, the patient's age, sex, and diet, the severity of any infection,time of adminstration and other clinical factors. The dosage may varywith the type of matrix used in the reconstitution and the types ofadditional proteins in the composition. The addition of other knowngrowth factors, such as IGF-I (insulin like growth factor I), to thefinal composition, may also affect the dosage.

[0092] Progress can be monitored by periodic assessment of cartilageformation, and/or repair. The progress can be monitored by methods knownin the art, for example, X-rays (CT), ultra-sound, MRI, arthroscopy andhistomorphometric determinations.

[0093] In another embodiment, as is exemplified in Example 1 theinvention provides a method of screening candidate nucleic acid sequencewhich is involved in the early stages of cartilage development, saidmethod comprising the step of: obtaining a cell; transfecting said cellwith a vector comprising a nucleic acid sequence encoding to FGFR3;obtaining mRNA from said cell; synthesizing cDNA from said mRNA;amplifying said cDNA-hybrid, so as to obtain an amplified product;detecting said amplified product; and comparing said amplified productsfrom said sample to amplified products derived from known samplesthereby identifying candidate nucleic acid sequence winch is involved inthe early stages of cartilage development.

[0094] The term “involved in the early stages of cartilage development”refers hereinabove to any gene, which is either upregulated ondownregulated during the stage of differentiation into a cartilage cell.Such genes will enable development of drugs which will ether enhance orsuppress cartilage formation or repair.

[0095] The step of “synthesizing” refer to step of building cDNAcomplementary to the mRNA template. As refer hereinabove and in theclaims section, the step of “amplifying” refer to the selectivereplication of a cDNA in greater number than usual. As refer hereinabove and in the claims section, the step of “separating” refer to thestep of separation of the products using for example, gelelectrophoresis. As refer hereinabove and in the claims section, thestep of “detecting” refer to the step of noticing, which is done, forexample by visualization of the amplified product's bands. As referhereinabove and in the claims section, the step of “comparing” refers tothe step of searching for differences between the amplified productsderived from the at least two samples. The term “RNA” refers to anoligonucleic in which the sugar is ribose, as opposed to deoxyribose inDNA. RNA is intended to include any nucleic acid, which can be entrappedby ribosomes and translated into protein. The term “mRNA” refers tomessenger RNA.

[0096] RNA can be extracted from cells or tissues according to methodsknown in the art. In a preferred embodiment, RNA can be extracted frommonolayers of mammalian cells grown in tissue culture, cells insuspension or from mammalian tissue. RNA can be extracted from suchsources by, e.g., treating the cells with proteinase K in the presenceof SDS. In another embodiment, RNA is extracted by organic solvents. Inyet another embodiment, RNA is extracted by differential precipitationto separate high molecular weight RNA from other nucleic acids. RNA canalso be extracted from a specific cellular compartment, e.g., nucleus orthe cytoplasm. In such methods, the nucleus is either isolated forpurification of RNA therefrom, or the nucleus is discarded forpurification of cytoplasmic RNA. Further details regarding these andother RNA extraction protocols are set forth, e.g., in Molecular CloningA Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis(Cold Spring Harbor Laboratory Press: 1989).

EXAMPLES

[0097] Experimental Procedures

[0098] DNA constructs and Transient Transfections

[0099] For the assessment of the transcriptional activity a dimmer ofthe double-stranded oligonucleotide of the Brachyury binding element(BBE) AATTTCACACCTAGGTGTGAAATT (Kispert et al., 1995) was incorporatedin the BamHI site before the HSV thymidine kinase minimal promoter fusedto the cloramphenicol acetyltransferase (CAT)-reporter of pBLCAT5(Boshalt et al., 1992) to give reporter plasmid pBBE-CAT5. 20 h beforetransfection, human embryonic kidney HEK293T cells were plated at adensity of 1×104 /cm2 in 6-well plates and allowed to grow under normalculture conditions. For co-transfection experiments, 250 ng per well ofBrachyury expression vector and 250, 500 or 750 ng of the expressionvector encoding dnBrachyury. Empty vector was added to adjust the amountof expression plasmids at 1 ug/ml. 260 ng of BBE-CAT reporter(pBBE-CAT5) was added in the presence of 140 ng of RSV-lacZ vector usingthe DOSPER procedure (see below). Cells were allowed to incubate for 48h. Then, cells were collected and b-galactosidase assays were performedwith the chemiluminescent b-gal reporter gene assay (Roche Diagnostics,Mannheim, Germany) and CAT-assays were carried out with the CAT ELISAkit (Roche Diagnostics, Mannheim, Germany). b-gal assay results wereused to normalize the CAT assay results for transfection efficiency. AllDNA transfection experiments were repeated at least three times intriplicate.

[0100] Cell Culture and Permanent Transfections

[0101] Human embryonic kidney cells HEK293T and murine C3H10T1/2progenitor cells were routinely cultured in tissue culture flasks inDulbecco's modified Eagle's medium supplemented with 10%heat-inactivated FCS, 0.2 nM L-glutarnine, and antibiotics (50 units/mlpenicillin, 50 mg/ml streptomycin). Cells were transfected using DOSPERaccording to the manufacturer's protocol (Roche Diagnostics, Mannheim,Germany). C3H10T1/2 cells which recombinantlly express BMP2(C3H10T1/2-BMP2) cells were obtained by co-transfection with pSV2pacfollowed by selection with puromycin (2.5 ug/ml). FGFR3, Brachyury andT-box domain were PCR-amplified and cloned into expression vectorspMT7T3 and pMT7T3-pgk vectors which are under the control of the LTR ofthe myeloproliferative virus or of the murine phosphoglycerate kinasepromoter-1, respectively (Ahrens et al., 1993). The integrity of theconstructs was confirmed by sequencing. HA-tags were carboxyterminallyadded to fill-length Brachyury and Brachyury's T-box domain by PCR withprimers encoding the respective peptide sequence. Stable expression ofthe DNA binding T-box domain (aa 1-229) and of the dominant-negativehuman FGFR3 without the cytoplasmatic tyrosine kinase domains (aa 1-414)in the C3H10T1/2-BMP2 background was done by co-transfection with pAG60,conferring resistance to G418 (750 ug/ml). Individual clones werepicked, propagated, and tested for recombinant FGFR3, dnFGFR3, Brachyuryor T-box domain (dnBrachyury) expression by RT-PCR (see below). Selectedcell clones were subcultivated in the presence of puromycine orpuromycine/G418 and the selective pressure was maintained duringsubsequent manipulations. C3H10T1/2 cells were cultured in DMEMcontaining 10% fetal bovine serumn. The features of C3H10T1/2-BMP2 cellshave been described (Ahrens et al., 1993; Hollnagel et al., 1997;Bächner et al., 1998). For the assessment of in vitroosteo-/chondrogeniic development, cells were plated at a density of57.5×103 cells/cm2. After reaching confluence (arbitrarily termed day 0)ascorbic acid (50 ug/ml) and 10 mM b-glycerophosphate were added asspecified by Owen et al., 1990 (1990).

[0102] BMP2 Inductions

[0103] For BMP2-stimulation studies, C3H10T1/2 cells were plated at adensity of 1×104 per cm² in a 9-cm culture dish. After 48 h cells werewashed 3× with PBS and then cells were starved for 24 h in DMEM withoutserum. Before induction the medium was replaced with fresh DMEM withoutserum. Cells were then treated for the indicated times using recombinantBMP2 from E. coli (50 ng/ml). Cycloheximide (50 ug/ml) treatment started30 min prior to the addition of BMP2.

[0104] RNA Preparation and RT-PCR

[0105] Total cellular RNAs were prepared by TriReagentLS according tothe manufacturer's protocol (Molecular Research Center Inc.). Five ug oftotal RNA was reverse transcribed and cDNA aliquots were subjected toPCR. RT-PCR was normalized by the transcriptional levels of HPRT. TheHPRT-specific 5′ and 3′ primers were GCTGGTGAAAAGGACCTCT andAAGTAGATGGCCACAGGACT, respectively. The following 5′ and 3′ primers wereused to evaluate osteo/chondrogenic differentiation: SEQ ID. No. 3:collagen 1a1: GCCCTGCCTGCTTCGTG, SEQ ID. No. 4: CGTAAGTTGGAATGGTTTTT;collagen 2a1: SEQ ID. No. 5: CCTGTCTGCTTCTTGTAAAAC, SEQ ID. No. 6:AGCATCTGTAGGGGTCTTCT; SEQ ID. No. 7: osteocalcin: GCAGACCTAGCAGACACCAT,SEQ ID. No. 8: GAGCTGCTGTGACATCCATAC; PTH/PTHrP-receptor: SEQ ID. No. 9:GTTGCCATCATATACTGTTTCTGC, SEQ ID. No. 10: GGCTTCTTGGTCCATCTGTCC; FGFR3:SEQ ID. No. 11: CCTGCGCAGTCCCCCAAAGAAG; SEQ ID. No. 12:CTGCAGGCATCAAAGGAGTAGT; FGFR2: SEQ ID. No. 13: TTGGAGGATGGGCCGGTGTGGTG,SEQ ID. No. 14: GCGCTTCATCTGCCTGGTCTTG. The primer pairs for Brachyuryand Sox9 have been described in (Johansson and Wiles, 1995) and(Zehentner et al., 1999), respectively. Vector-borne transcripts forBrachyury were evaluated with nested primers sets with either vectorspecific 5′- or 3′-primers: SEQ ID. No. 15: TTAGTCTTTTTGTCTTTTATTTCA;SEQ ID. No. 16: GATCGAAGCTCAATTAACCCTCAC.

[0106] Western Blotting

[0107] Recombinant cells from petri dishes (13.6 cm diameter) wereharvested at different time points before (day B2), at (day 0) and after(days 2, 4, 7) confluence. Lysis was in RIPA buffer (1% (v/v) nonidetP-40, 0.1% SDS (w/v), 0.5% sodium deoxycholate in PBS, containing 100ug/ml PMSF, 2 ug/mil aprotinin, and 1 mM Na3VO4). Lysates werecentrifuged (30 min, 10.000 g, 4C.) and the supernatants were stored at−70C. until analysis. Protein concentration of the lysates wasdetermined using coomassie brilliant blue. Protein was precipitated withethanol, resuspended in reducing (containing DTT) or non-reducing samplebuffer and subjected to SDS-gel electrophoresis in 12.5% Tpolyacrylamide gels (20 ug/lane). Proteins were transferred tonitrocellulose membranes by semidry-blotting. Protein transfer waschecked by staining of the membranes with Ponceau S. After blocking,membranes were incubated incubated overnight at 4 C. with a polyclonalantibody to the HA-tag (SC-805, Santa Cruz Biotechnology, Santa Cruz,Calif.) diluted 1:200 in blocking solution. FGFR3 and FGFR2 antibodieswere from Santa Cruz Biotechnology (#SC-123, #SC-122; Santa Cruz,Calif.). The secondary antibody (Dianova, Hamburg) was applied at 1:5000in blocking solution for 2 h at room temperature. Color development wasperformed with 4-chloro-1-naphthol and H2O2.

[0108] Histological Methods and Verification of Cellular Phenotypes

[0109] Osteoblasts exhibit stellate morphology displaying high levels ofalkaline phosphatase, which was visualized by cellular staining withSIGMA FAST BCIP/NBT (Sigma, St. Louis, Mo.). Proteoglycan secretingchondrocytes were identified by staining with Alcian Blue at pH 2.5 andstaining with Safranin O (Sigma, St. Louis, Mo.). Forcollagen-immunohistochemistry cells were washed with PBS and fixed withmethanol for 15 min at −20 C. by methanol. Primary antibodies werediluted with 1% goat serum in PBS. Monoclonal anti-collagen IIantibodies (Quartett Immunodiagnostika, Berlin, Germany, #031502101)were diluted 1:50 and monoclonal anti-collagen X antibodies (QuartettImmunodiagnostika, Berlin, Germany, #031501005) 1:10, respectively.Incubation was for 1 hour at room temperature followed by staining withZymed HistoStain SP kit (Zymed Laboratories Inc., San Francisco, Calif.)applying the manufacturer's protocol. A positive signal is indicated bya red color precipitate of AEC (aminnoetlhylcarbazole).

[0110] In Vivo Transplantation

[0111] Before in vivo transplantation, aliquots of 2-3×106 cells weremounted on individual type I collagen sponges (Colastat7 #CP-3n,Vitaphore Corp., 2×2×4 mm.) and transplanted into the abdominal muscleof female nude mice (4-8 weeks old). Before transplantation animals wereanesthetized with ketamine-xylazine mixture 30 ul/per mouse i.p. andinjected i.p. with 5 mg/mouse of Cefamzolin (Cefamezin7, TEVA). Skin wasswabbed with chlorhexidine gluconate 0.5%, cut in the mijddle abdominalarea, an intramuscular pocket was formed in a rectal abdominal muscleand filled with the collagen sponge containing cells. Skin was suturedwith surgical clips. For the detection of engrafted C3H10T1/2 cells themice were sacrificed 10 days and at 20 days after transplantation.Operated transplants were fixed in 4% paraformaldehyde cryoprotectedwith 5% sucrose overnight, embedded, and frozen. Sections were preparedwith a cryostat (Bright, model OTF) and stained with H&E, Alcian Blueand Safranin O.

[0112] RNA-In Situ-Hybridization

[0113] Embryos were isolated from pregnant NMRI mice at day 18.5 postconceptionem (dpc). The embryos were fixed overnight with 4%parafonnaldehyde in PBS at 4C. 10 um cryosections were mounted onaminopropyltrimethoxysilane coated slides and non-radioactive RNA-insitu-hybridizations were done as described (Bachner et al., 1998) and byfollowing the instructions of the manufacturer (Roche, Mannheim). Inshort: For hybridization sense and antisense RNA probes from a 1.8 kbmurine Brachyury cDNA was used. For the generation of collagen 1a1 orcollagen 2a1 the vector pMT7T3 was used harbouring specific probes(Metsäranta et al., 1991). Hybridization was performed with 0.5-2 ugdenatured riboprobe/ml) over night at 65C. in a humid chamber. Fordigoxygenin (DIG)-detection slides were blocked in 5×SSC, 0.1% Triton,20% FCS for 30 minutes following two washes with DIG-buffer 1 (100 mMTris, 150 mM NaCl, pH 7.6) for 10 minutes. Slides were incubated inanti-DIG-alkaline phosphatase coupled antibodies diluted 1:500 inDIG-buffer 1 over night in a humid chamber. Slides were washes with 0.1%Triton in DIG-buffer 1 for 2 hours with several changes of the washingsolution and equilibrated in DIG-buffer 2 (100 mM Tris, 100 mM NaCl, 50mM MgCl2). Detection was performed using BM-purple substrate (Roche,Mannheim) in DIG-buffer 2 with 1 mM Levamisole for 1-6 hours dependingon the probe. Reaction was stopped in TE-buffer and slides wereincubated in 3% paraformaldelhyde in PBS for 3 minutes, in 0.1 M glycinein PBS for 3 minutes and washed three times in PBS for 3 minutes. Slideswere counterstained with 0.5% methylenegreen in PBS for 1 minute,dehydrated in graded alcohol series, air-dried and mounted with Eukitt.

[0114] Experimental Results

Example 1

[0115] BMP2-Dependent Chondrogenic Development in C3H10T1/2 MSCsInvolves FGF-Receptor 3.

[0116] During a substractive screen for BMP-regulated genes inrecombinant BMP2-expressing C3H10T1/2 (C3H10T1/2-BMP2) cellsupregulation of the Fibroblast Growth Factor Receptors 3 and 2 (FGFR3,FGFR2) was noted at both the transcriptional and protein levels (FIGS.1a, c, respectively). These two receptor types exhibit differentinduction kinetics. FGFR3 is upregulated during early stages ofcultivation in the stable C3H10T1/2-BMP2 line while FGFR2 shows adelayed response (FIGS. 1a, c). The fast upregulation of FGFR3 seems tobe due to an immediate response to BMP2 since exogenously-added BMP2mediated FGFR3 transcription in wild-type C3H10T1/2 cells in thepresence of cycloheximide (FIG. 1b). In contrast to FGFR3 and FGFR2 isFGFR1 constitutivelly expressed in wild type and C3H10T1/2-BMP2 cells(FIG. 1a) while FGFR4 does not show any significant rates of expression.Since FGFs and their receptors are crucial modulators of chondrogenicdevelopment, an assessment of whether the immediate BMP2-dependentupregulation of FGFR3 in C3H10T1/2 is involved in the onset ofchondrogenic differentiation was conducted. Indeed the resultsdemonstrated that forced expression of the wild-type FGFR3 (FGFR3WT) wassufficient for the development of morphologically distinct chondrocytesin C3H10T1/2-FGFR3WT cells (FIGS. 1d, e). Moreover, the constitutivelyactive mutant FGFR3 (Ach, G380R) possesses the same capacity. The forcedexpression of FGFR3WT in MSCs stimulates MAPK signaling in these cellsas documented by enhanced levels of ERK1 and ERK2 phosphorylation (rightpanels in FIG. 1d), leads to the development of histologically distinctchondrocytes and induces or increases expression of chondrogenic markergenes such as collagen 2a1, the PTH/PTHrP receptor and transcriptionfactor Sox9 (FIG. 1e).

[0117] The immediate BMP2-dependent upregulation of FGFR3 in MSCs(C3H10T1/2) and the inherent capacity of this receptor to initiatechondrogenic development in these cells prompted a screen forFGFR3-regulated transcription factors. It was observed that among thetranscription factors tested, the T-box transcription factor Brachyurywas upregulated in FGFR3-expressing C3H10T1/2 cells (see also FIG. 5a).Thereupon, the chondrogenic potential that Brachyury possesses afterrecombinant expression in wild-type C3H10T1/2 cells (see below) wasdemonstrated.

Example 2

[0118] Forced Expression of the T-box Factor Brachyury Leads toChondrogenic Development in C3H10T1/2 Mesenchymal Stem Cells.

[0119] Brachyury has originally been described as the first member of afamily of transcription factors that harbors a T-box as the DNA-bindingdomain. In order to assess that the FGFR3-dependent upregulation of theT-box factor Brachyury in C3H10T1/2 plays a role in chondrogenesisBrachyury cDNA was expressed under the control of the murinephosphoglycerate kinase-1 (PGK-1) in mesenchymal stem cell lineC3H10T1/2 to allow moderate expression levels of Brachyury(C3H10T1/2-Brachyury). The recombinant expression of Brachyury cDNAunder the control of the murine phosphoglycerate kinase-1 (PGK-1) inMSCs (FIG. 2a) gave uise to efficient chondrogenic differentiationresulting in alkaline phosphatase positive cells (beginning at day 4)and Alcian Blue positive chondrocyte-like cells (at day 10post-confluence; FIG. 2b). Three individual C3H10T1/2 clones wereinvestigated in regard to their chondrogenic potential and gave similarresults. Immunohistochemistry confirms the presence of thechondrocyte-specific collagen 2 but not of collagen X which is typicalfor late stages of chondrocytic differentiation (hypertophicchondrocytes) (FIG. 2b, left panel). Major marker genes of chondrogenicand, also, of osteogenic development show a transient (collagen 2a1,PTH/PTHrP-receptor) or permanent upregulation (osteocalcin gene and thechondrogenic transcription factor Sox9) in C3H10T1/2-Brachyury incomparison with C3H10T1/2 cells which were stably transfected with anempty expression vector (FIG. 2c). Although, the induction of theosteocalcin gene indicates an osteogenic potential forC3H10T1/2-Brachyury, ectopic transplantation of these cells in murineintramuscular sites results exclusively in the massive formation ofproliferating chondrocytes and cartilage (FIG. 2d). These ectopictransplantations have been performed three times and in all cases thesetransplants developed chondrocytes and cartilage. After both 10 and 20days transplants exhibit a histological presence of proteoglycans(Alcian Blue, Safranin O) while bony elements or mineralized particlesare not observed (FIG. 2d). After 20 days the ectopic implants showareas of extensive extracellular matrix production as visualized byhistological analyses (FIG. 2d). The use of stronger viral promoterssuch as the LTR of the myeloproliferative virus (MATERIALS and METHODS)resulted in increased cellular proliferation without the apparentformation of histologically distinct mesenchymal cell types.

Example 3

[0120] Dominant-Negative Brachyury Interferes with BMP2-DependentChondrogenic Development in MSCs.

[0121] A partial nuclear localization signal (NLS) which has beenattributed to the T-box domain should allow a substantial nuclearaccumulation (Kispert et al., 1995). The dominant-negative nature of theT-box domain was confirmed in DNA co-transfection assays performed inHEK293 T cells. This cell line does not express Brachyury. ExogenousBrachyury transactivated a construct containing two copies of theconsensus Brachyury binding element (BBE) oligonucleotide fused to aminimal HSV thymidine kinase (TK)-minimal promoter-CAT chimeric gene,pBBE(2×)-CAT5 (FIG. 3a). Indeed, co-transfection of pBBE(2×)-CAT5 with arecombinant Brachyury-expressing vector resulted in a 25-foldactivation, whereas an empty expression vector had no effect (FIG..3 a).Co-transfection of full-length Brachyury (Brachyury wt) with increasingamounts of an expression vector expressing the T-box domain(dnBrachyury) (1:1, 1:2, 1:3) led to a clear decrease in CAT (7-fold).Exogenous dnBrachyury alone transactivated pBBE(2×)-CAT5 (BBE) only3-fold.

[0122] The forced expression of tie HA-tagged T-box domain (dnBrachyury)is observed throughout in vitro cultivation (FIG. 3b) and stronglyinterfered with the BMP2-mediated formation of alkaline phosphatasepositive osteoblast-like and Alcian Blue chondrocyte-like cells in vitro(FIG. 3e). In vivo, in ectopic transplantations of C3H10T1/2-BMP2 inintramuscular sites, dnBrachyury try allowed the development ofconnective tissue only (FIG. 3e). In addition, the chondrocyte-specificcollagen 2a1 mRNA levels are more sensitive to the presence ofdnBrachyury than mRNA levels of the distinct osteogenic markerosteocalcin. The latter is hardly affected, consistent with the ideathat Brachyury possesses a predominant chondrogenic capacity in thisparticular cell type. Interestingly, the BMP2-mediated transcriptionalupregulation of FGFR3 in C3H10T1/2 is not obstructed by dnBrachyuryindicating that the immediate BMP2-mediated FGFR3 induction isindependent of Brachyury or other T-Box factors (FIGS. 3c, d). However,FGFR2-expression that bits a delayed response in C3H10T1/2-BMP2 cells(FIGS. 1 and 3) displays a high sensitivity to dnBrachyury. BMP-mediatedFGFR2 expression is almost completely suppressed by thedominant-negative acting T-box domain (FIGS. 3c, d). This may indicatethat that the presence of FGFR2 seems necessary for theosteo-/chondrogenic differentiation in this mesencehymal progenitor line(FIGS. 3c,d).

[0123] Furthermore, this suggests a hierarchy of FGFR-mediated signalingfor chondrogenic development. FGFR3-dependent signaling is induced atfirst by BMP2 and as a consequence, FGFR2 mediated signaling becomesactive. Such a model is proposed in FIG. 7. This model predicts that aforced expression of dominant-negative FGFR3 would interfere withBMP2-mediated chondrogenesis and with FGFR2 and Brachyury expression.Indeed, an FGFR3-variant without the cytoplasmatic tyrosine-kinasedomains dowregulates BMP2-dependent mRNA expression levels of FGFR2 andBrachyury (FIG. 4b) and interferes with the histological manifestationof alkaline phosphatase or Alcian Blue positive chondrocyte like cells(FIG. 4a).

Example 4

[0124] FGFR3 and the T-box Factor Brachyury are Involved in anAutoregulatory Loop for Chondrogenic Development in C3H10T1/2Progenitors

[0125] During amphibian gastrulation, mesodermal Brachyury is involvedin an autoregulatory loop with FGF that is present in the embryo (1998).In C3H10T1/2 cells several FGF genes tested (FGF2, 4, and 9) were notBrachyury- or FGFR3-regulated and, therefore, are unlikely members ofsuch a loop. However, a loop seems to exist between FGFR3 and Brachyurysince forced expression of either one lead to the induction of the otherone in C3H10T1/2 (FIG. 5a). These experiments indicate that afterBMP2-mediated initiation of the chondrogenic lineage, the chondrogenicdifferentiation may advance for some time in a BMP2-independent fashionmaintained by the autoregulatory loop between FGFRs and FGF-regulatedtranscription factors such as the T-box factor Brachyury.

[0126] In a preceding study it was shown that BMP-mediated R-Smadsignaling alone is not sufficient for cartilage development in C3H10T1/2cells. Thereby, forced expression of Smad1 or the biologically activeSmad1-MH2 domain is able to mimic BMP2-mediated onset of osteogenicdifferentiation (Takeuchi et al., 2000). However, in contrast toosteogenic marker genes such as the osteocalcin gene, Smad1-MH2domain-signaling is not sufficient to mimic BMP2-dependent FGFR3—and theconcomitant Brachyury-gene induction (FIG. 5b). Other BMP-activatedR-Smads such as Smad5 and Smad8 are also unable to mediate or to mimicBMP2-dependent FGFR3-induction in C3H10T1/2 cells indicatingR-Smad-MH2-independent pathways for FGFR3 induction or, alternatively,cooperative activities of R-Smads with other transcription factors(Mazars et al., 2000).

Example 5

[0127] The T-box Factor Brachyury is Expressed in Maturing CartilageDuring Murine Embryonic Development

[0128] Brachyury is expressed at high levels early in vertebrateembryonic development and is involved in gastrulation and in thedose-dependent determination of mesodermal cell fates. Aftergastrulation, Brachyury-expression is downregulated and persists in thenotochord to the end of embryogenesis (Kispert and Herrmann, 1994).Comparative mRNA expression analysis of murine Brachyury (Bra), collagen1a1 (co1 1a1) and collagen 2a1 (co1 2a1) in skeletal development (18.5dpc) indicates that Brachyury is expressed at significant levels incartilage forming cells of the intervertebral disks and in limb buddevelopment (FIG. 6). Expression of Brachyury is enhanced inintervertebral disc development in the nucleus pulposus in 18.5 dpcmouse embryos (FIGS. 6A, a, d) confirming earlier reports (Wilkinson etal., 1990). Collagen 1a1 is expressed in the outer annulus (arrowheadsin FIGS. 6A b, e), and collagen 2a1 in the cartilage primordium of thevertebrae (FIGS. 6A, c, f). In transversal sections made at the level ofthe upper lumbar vertebra, expression of Brachyury is in additiondetectable in distinct chondrogeric cells of the neural arch (FIGS. 6A,h) whereas collagen 1a1 expression is maintained in the outer annulus(FIGS. 6A, i), as is collagen 2a1 in the cartilage primordium (FIGS. 6A,j). In murine limb bud development (18.5 dpc; hind limb) expression ofBrachyury is evident in distinct chondrogenic cells of the formingmetatarsal bones (FIGS. 6B, a-c). In contrast, collagen 1a1 is expressedin the outer periosteal layer (FIGS. 6B, d-f) and collagen 2a1expression is enhanced in differentiating chondrocytes (FIGS. 6B, g-i).Interestingly, like in intervertebral disc formation, the expression ofBrachyury is only evident in chondrocyte-like cells that do not expressCo1 2a1 indicating that Brachyury expression is upregulated inchondrogenic cells before or after collagen 2 expressions.

Example 6

[0129] The T-box Factor Brachyury is Expressed in Human AdultMesenchymal Cells Following Transfection with Brachyury Plasmid.

[0130] Cells Isolation:

[0131] Human Adult Mesenchymal Stem Cells (hAMSCs) were isolated fromexplants of human bone marrow surgical waste and expanded in vitro.Isolation of hMSCs was performed as follows: 10 ml marrow aspirates werecollected into a tube with 6000 U heparin, washed with PBS, andrecovered cells were collected by centrifugation at 900 g. Collectedcells were then loaded onto Percoll solution (density 1.073 g/ml). Cellseparation was accomplished by centrifugation at 1100 g (30 min at 20uC). Nucleated cells collected were washed twice with PBS and thencultured in 100 mm culture plates.

[0132] Tissue Culture:

[0133] Cells were cultured in low glucose, low bicarbonate DMEM medium(Beit Haemek)+10% fetal calf serum (Biet Haemek), the enviromentalconditions were of 5% CO2 and 370 C.

[0134] Cells Transfection:

[0135] 3×10⁶ hAMSCs were transfected with 30 ug of the Brachyury plasmidusing the Amaxa Nucleofector™ technology and in accordance with themanufacturer's preliminary protocol for hAMSCs. Briefly, the harvestedcells were aliquoted in 5×10⁵ cells, recovered by centrifugation, andre-suspended in 100 μl of Amaxa's nucleofection solution. Five microgramof DNA plasmid were added to the suspended cells, mixed well andtransferred to electroporation cuvette provided by the Amaxanucleofection kit. The electroporation was performed using 5 differentprograms (U28, C12, C17, E14, G22) that basically differ in theintensity and the length of the electric pulse. Immediately after theelectroporation, the cells were transferred into 6-well plates,containing 4ml complete growth medium equilibrated to 37C., 5% CO2, andincubated at 37° C. in 5% CO₂ atmosphere for 24 hours.

[0136] Detection of Gene Expression:

[0137] 24 hours post transfection RNA was isolated from the cells usingthe Trizol reagent and protocol provided by the manufacturer (LifeSciences). 2 ug of RNA were transformed into cDNA by ReverseTranscriptase (RT) reaction. PCR was then performed using specificprimers to the Brachyury cDNA. 20 ul of the PCR reaction sample wereloaded into a 2% Agarose gel stained with Etidium Bromide. The gelanalysis demonstrated a band matching the expected amplified region inthe Brachyury cDNA (see FIG. 8).

What is claimed is:
 1. A method of enhancing repair of a cartilagecomprising the step of administering to a subject an effective amount ofa cell which expresses at least one factor of the T-box family, therebyenhancing repair of the cartilage.
 2. The method of claim 1, whereinsaid cell is a mesenchymal stem cell, a progenitor cell, a cell iscapable to differentiate into a chondrocyte a fibroblast or a synovialcell.
 3. The method of claim 1, wherein said factor of the T-box isbrachyury.
 4. The method of claim 1, wherein said cell further expressesfactor which upregulates the expression of the T-box transcriptionfactor.
 5. The method of claim 4, wherein said factor which upregulatesthe expression of the factor of the T-box is FGF or BMP2.
 6. A method ofinducing formation of a cartilage comprising the step of administeringto a subject an effective amount of a cell which expresses at least onefactor of the T-box family, thereby inducing formation of the cartilage.7. The method of claim 6, wherein said cell is a mesenchymal stem cell,a progenitor cell, a cell is capable to differentiate into a chondrocytea fibroblast or a synovial cell.
 8. The method of claim 6, wherein saidfactor of the T-box is brachyury.
 9. The method of claim 6, wherein saidcell further expresses factor which upregulates the expression of theT-box transcription factor.
 10. The method of claim 9, wherein saidfactor which upregulates the expression of the factor of the T-box isFGF or BMP2.
 11. A method of enhancing repair of a cartilage in the bodycomprising the step of administrating a recombinant vector whichcomprises a nucleic acid encoding a factor of the T-box family to thecartilage of a subject, thereby enhancing repair of the cartilage. 12.The method of claim 1 1, wherein said factor of the T-box is brachyury.13. The method of claim 11, wherein said method further comprisesadministering a recombinant vector which comprises a nucleic acidencoding a factor which upregulates the expression of the T-boxtranscription factor.
 14. The method of claim 13, wherein said factorwhich upregulates the expression of the factor of the T-box is FGF orBMP2.
 15. A method of inducing formation of a cartilage in the bodycomprising the step of administrating a recombinant vector whichcomprises a nucleic acid encoding a factor of the T-box family to thecartilage of a subject, thereby inducing formation of the cartilage. 16.The method of claim 15, wherein said factor of the T-box is brachyury.17. The method of claim 15, wherein said method further comprisesadministering a recombinant vector which comprises a nucleic acidencoding a factor which upregulates the expression of the T-boxtranscription factor.
 18. The method of claim 17, wherein said factorwhich upregulates the expression of the factor of the T-box is FGF orBMP2.
 19. A method of inducing chondrocyte differentiation comprisingthe step of administering of a recombinant vector which comprises anucleic acid encoding a factor of the T-box family, thereby inducingchondrocyte formation.
 20. The method of claim 19, wherein said factorof the T-box is brachyury.
 21. The method of claim 19, wherein saidmethod further comprises administering a recombinant vector whichcomprises a nucleic acid encoding a factor which upregulates theexpression of the T-box transcription factor.
 22. The method of claim19, wherein said factor which upregulates the expression of the factorof the T-box is FGF or BMP2.
 23. A method of repairing or forming acartilage in a subject in need comprising the steps of: obtaining a cellfrom of the subject; transfecting said cell with a recombinant vectorcomprising a nucleic acid sequence encoding a factor of the T-boxfamily, so as to obtain an engineered cell which expresses a factor ofthe T-box family; and administering said engineered cell to the subject.24. The method of claim 23, wherein said cell is a mesenchymal stemcell, a progenitor cell, a cell is capable to differentiate into achondrocyte a fibroblast or a synovial cell.
 25. The method of claim 23,wherein said factor of the T-box is brachyury.
 26. The method of claim23, wherein said cell further expresses factor which upregulates theexpression of the T-box transcription factor.
 27. The method of claim26, wherein said factor which upregulates the expression of the factorof the T-box is FGF or BMP2.
 28. The method of claim 23, wherein saidmethod further comprises administering a recombinant vector whichcomprises a nucleic acid encoding a factor which upregulates theexpression of the T-box transcription factor.
 29. The method of claim28, wherein said factor which upregulates the expression of the factorof the T-box is FGF or BMP2.
 30. A method for the production oftransplantable cartilage matrix, the method comprising the steps of:obtaining a cell; transfecting said cell with a recombinant vectorcomprising a nucleic acid sequence encoding a factor of the T-boxfamily, so as to obtain an engineered cell which expresses a factor ofthe T-box family; and culturing said cell with the cell-associatedmatrix for a time effective for allowing formation of a transplantablecartilage matrix.
 31. The method of claim 30, wherein said cell is amesenchymal stem cell, a progenitor cell, a cell is capable todifferentiate into a chondrocyte a fibroblast or a synovial cell. 32.The method of claim 30, wherein said factor of the T-box is brachyury.33. The method of claim 30, wherein said cell further expresses factorwhich upregulates the expression of the T-box transcription factor. 34.The method of claim 33, wherein said factor which upregulates theexpression of the factor of the T-box is FGF or BMP2.
 35. The method ofclaim 30, wherein said method further comprises administering arecombinant vector which comprises a nucleic acid encoding a factorwhich upregulates the expression of the T-box transcription factor. 36.The method of claim 35, wherein said factor which upregulates theexpression of the factor of the T-box is FGF or BMP2.
 37. An engineeredcell which expresses a factor of the T-box family.
 38. The cell of claim37, wherein said cell is a mesenchymal stem cell, a progenitor cell, acell is capable to differentiate into a chondrocyte a fibroblast or asynovial cell.
 39. The cell of claim 37, wherein said factor of theT-box is brachyury.
 40. The cell of claim 37, wherein said cell furtherexpresses factor which upregulates the expression of the T-boxtranscription factor.
 41. The cell of claim 40, wherein said factorwhich upregulates the expression of the factor of the T-box is FGF orBMP2.
 42. The cell of claim 37, wherein said cell is a human mesenchymalcell.
 43. A composition comprising an engineered cell which expresses afactor of the T-box family and a pharmaceutically acceptable carrier.44. The composition of claim 43, wherein said cell is a mesenchymal stemcell, a progenitor cell, a cell is capable to differentiate into achondrocyte a fibroblast or a syniovial cell.
 45. The composition ofclaim 43, wherein said factor of the T-box is brachyury.
 46. Thecomposition of claim 43, wherein said cell further expresses factorwhich upregulates the expression of the T-box transcription factor. 47.The composition of claim 46, wherein said factor which upregulates theexpression of the factor of the T-box is FGF or BMP2.
 48. Thecomposition of claim 46, wherein said composition is a pharmaceuticallycomposition.
 49. A composition comprising at least one recombinantvector which comprises a nucleic acid sequence encoding at least onefactor of the T-box family and a pharmaceutically acceptable carrier.50. The composition of claim 49, wherein said composition is apharmaceutically composition.
 51. The composition of claim 49, whereinsaid factor of the T-box is brachyury.
 52. The composition of claim 49,wherein said method further comprises administering a recombinant vectorwhich comprises a nucleic acid encoding a factor which upregulates theexpression of the T-box transcription factor.
 53. The composition ofclaim 52, wherein said factor which upregulates the expression of thefactor of the T-box is FGF or BMP2.
 54. An implant device comprising atleast one engineered cell which expresses a factor of the T-box familyand a pharmaceutically acceptable carrier.
 55. The device of claim 54,wherein said cell is a mesenchymal stem cell, a progenitor cell, a cellis capable to differentiate into a chondrocyte a fibroblast or asynovial cell.
 56. The method of claim 54, wherein said factor of theT-box is brachyury.
 57. The method of claim 54, wherein said cellfurther expresses factor which upregulates the expression of the T-boxtranscription factor.
 58. The method of claim 57, wherein said factorwhich upregulates the expression of the factor of the T-box is FGF orBMP2.
 59. The method of claim 54, wherein said cell is human cell.
 60. Amethod of suppressing cartilage formation, comprising the step ofadministering to a subject in need an antagonist to a factor of theT-box family thereby suppressing cartilage formation.
 61. The method ofclaim 60, wherein said antagonist is an antibody, an antisense, aprotein, a nucleic acid or a carbonhydrate.
 62. The method of claim 60,wherein said antagonist is a dominant negative factor of the T-boxfamily.
 63. The method of claim 60, wherein said antagonist is adominant negative Brachyury.
 64. A method of screening candidate nucleicacid sequence, which is, involved in the early stages of cartilagedevelopment, said methods comprising the step of obtaining a cell;transfecting said cell with a vector comprising a nucleic acid sequenceencoding to FGFR3; obtaining mRNA from said cell; synthesizing cDNA fromsaid mRNA; amplifying said cDNA-hybrid, so as to obtain an amplifiedproduct; detecting said amplified product; and comparing said amplifiedproducts from said sample to amplified products derived from knownsamples thereby identifying candidate nucleic acid sequence which isinvolved in the early stages of cartilage development.
 65. The cell ofclaim 64, wherein said cell is a mesenchymal stem cell, a progenitorcell, a cell is capable to differentiate into a chondrocyte a fibroblastor a synovial cell.
 66. The cell of claim 64, wherein said cell is ahuman mesenchymal stem cell.